Fish and Shellfish Immunology 86 (2019) 93–100 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Cloning of six serpin genes and their responses to GCRV infection in grass T carp (Ctenopharyngodon idella) ∗∗ Liangming Chena,b, Rong Huanga, , Denghui Zhua,b, Yumeng Wangc, Rumana Mehjabina,b, ∗ Yongming Lia, Lanjie Liaoa, Libo Hea, Zuoyan Zhua, Yaping Wanga, a State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China b University of Chinese Academy of Sciences, Beijing, 100049, China c Wuhan University, Wuhan, 430072, China ARTICLE INFO ABSTRACT Keywords: Grass carp, an economically important aquaculture fish, is very sensitive to Grass Carp Reovirus (GCRV). Grass carp Haemorrhagic disease caused by GCRV infection can cause large-scale death of first-year grass carp, thereby Grass carp reovirus (GCRV) severely restricting the intensive culture. Serpins (serine protease inhibitors) belong to the protease inhibitor Serpins (serine protease inhibitors) gene family and are involved in numerous physiological and pathological processes, particularly coagulation and Coagulation and anticoagulation anticoagulation. Reports on grass carp serpins are scarce. Thus, we cloned six grass carp serpin genes (serpinb1, serpinc1, serpind1, serpinf1, serpinf2b and serping1) in this study. Molecular evolution showed that serpins be- tween grass carp and zebrafish or carp are the closest relatives. SERPIN domains in these6 serpins and reactive centre loop (RCL) along with their cleavage sites of 5 serpins (serpinb1, serpinc1, serpind1, serpinf2b and serping1) were predicted. Real-time quantitative PCR (RT-qPCR) showed that these serpins displayed tissue significance. Among them, serpinc1, serpind1, serpinf2b and serping1 had the highest expression levels in the liver. After GCRV infection, RT-qPCR showed that the liver-enriched serpins were significantly changed. Key procoagulant factor genes (kng-1, f2, f3a, f3b and f7) and anticoagulant genes (tpa, plg, thbd, proc and pros) also showed significant changes on the mRNA level. Comprehensive comparative analysis showed that the up-regulated expression of key clotting factor genes was more prominent than that of main anti-coagulation factor genes. Thus, the function of coagulation may be more dominant in grass carp during the GCRV infection, which may cause overproduction of thrombi. The serpins were involved in GCRV infection and liver-enriched serpins participate in the interaction between coagulation and anticoagulation. This study provided new insights into further research on the bio- logical functions of grass carp serpins and clarifying the molecular mechanism of GCRV affecting the homeostasis of grass carp blood environment. 1. Introduction have been identified in many species, including animals, plants, viruses, bacteria and archaea [6]. In normal living organisms, numerous proteases and their corre- A typical serpin protein has a conserved SERPIN domain that con- sponding protease inhibitors restrict each other and are in a state of tains three β-sheets (β-sheet A, β-sheet B and β-sheet C), 7-9 α-helices dynamic equilibrium [1]. In 1894, Fermi and Pernossi et al. discovered and a reactive centre loop (RCL) [7,8]. RCL serves as a functional region the first serpin (serine protease inhibitor) in human blood. Shultze et al. responsible for the interaction with the target enzyme, and its sequence isolated this serpin gene and named it α1PI in 1995 [2–4]. Hunt and determines serpin specificity [6]. In the proteolysis process, serpin Dayhoff subsequently compared the sequence similarities between proteins inhibit the proteolytic activity of serine/cysteine proteases α1PI, ovalbumin and ATIII and finally demonstrated the existence of through a “suicide” mechanism. The cleavage site of the RCL in the the serpin gene [5]. According to the similarity between different serpin serpin protein is then recognised, bound and cleaved by the corre- genes, the serpins are divided into 16 clades [6], which constitute a sponding target protease [8]. The terminal amino acid of RCL is in- large family of serine protease inhibitors. More than 3000 serpin genes serted into the β-sheet A, which is called a transition from a “stressed ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (R. Huang), [email protected] (Y. Wang). https://doi.org/10.1016/j.fsi.2018.11.008 Received 16 June 2018; Received in revised form 29 September 2018; Accepted 3 November 2018 Available online 12 November 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved. L. Chen et al. Fish and Shellfish Immunology 86 (2019) 93–100 (S) to relaxed (R),” and leads to the formation of an irreversible cova- expressed genes (including the serpin genes) in the four tissues (gill, lent complex between the target enzyme and the serpins, resulting in liver, spleen and intestine) are mainly concentrated in the “complement their simultaneous inactivation; thus, their corresponding biological and coagulation” cascade signalling pathway [27]. Abnormal blood functions are realised [9,10]. Serpin proteins involve in multiple basic clotting can cause pathological bleeding and thrombosis in organisms biology processes, including blood coagulation, complement activation, [12]. Thus, grass carp haemorrhagic disease caused by GCRV may be fibrinolysis, angiogenesis, inflammation and tumour suppression by related to coagulation abnormalities. inhibiting the activity of serine proteases (SPs) or cysteine proteases Serpins are among the most widely studied protease inhibitors [28]. (CPs) [7]. However, the serpins in grass carp are rarely studied. Only the biolo- The balancing process between blood coagulation and antic- gical function of serpinf1 in fatty inflammation has been studied in oagulation is the key to the normal body maintenance of blood flow and grass carp [29]. The cloning and expression analysis of these 6 serpins in prevention of blood loss [11]. Coagulation and anticoagulant factors grass carp facilitate the further research on the structure and related can restrain each other to ensure that the coagulation and antic- biological functions of the serpin gene family in grass carp and may oagulation process are in a dynamic equilibrium [11–13]. The normal provide new insights into the molecular mechanism of GCRV affecting blood clotting process, which eventually causes the change in the blood the homeostasis of blood environment in grass carp. from a liquid to a colloidal state, involves the formation of prothrombin activators, thrombin and fibrin [12,14]. According to the types of dif- 2. Materials and methods ferent coagulation factors, the process of coagulation cascade is divided into the intrinsic pathway (also known as contact activation pathway) 2.1. Experimental fish and the extrinsic pathway (also known as tissue factor pathway) [15]. The high molecular weight kininogen (HMWK), derived from Kini- Six-month-old grass carp (15 ± 3 cm, 40 ± 10 g) for gene cloning nogen-1 (KNG-1, encoded by kng-1), f12 and prekallikrein, are key and GCRV infection experiments were provided by the Guanqiao molecules that initiate the intrinsic pathway [16,17]. When f12 is ac- Experimental Base of the Institute of Hydrobiology, Chinese Academy tivated to f12a, it further converts f11 to f11a. Then, f11a activates f9 to of Sciences. Prior to the grass carp GCRV infection experiment, the form f9a. The f9a forms a tenase complex with f8a, which further ac- experimental fish were placed in a 500 L circulating water system at tivates f10 to form f10a [18]. In the extrinsic pathway, the tissue factor 28 °C with adequate dissolved oxygen, penicillin (100 U/ml) and (TF, also known as f3) forms a complex with f7a. This complex can streptomycin (100 μg/ml). Normal feeding was conducted for 1 week, activate f9 and f10; then, f9a further promotes f10 activation to form and experimental fish without abnormal conditions were used for f10a [15,19]. Finally, f10a and its cofactor f5a from both endogenous subsequent experiments. The absence of abnormalities on the one hand and exogenous pathways form a prothrombinase, which activates pro- referred to no abnormalities in outward appearance, feeding and ac- thrombin (f2) to thrombin (f2a) [18]. Thrombin transforms fibrinogen tivity; on the other hand, we used the PCR method to detect the GCRV into fibrin, and the blood clot formed by fibrin polymerisation exerts and found no GCRV infection. coagulation function [18]. Most of the coagulation factors involved in blood coagulation in the 2.2. Cloning of full-length cDNA from grass carp serpins living body are serine proteases, such as f2a, f5a, f7a, f8a, f9a, f10a and f11a [20]. Serine proteases are also involved in the anticoagulant Respectively, the homologous serpin sequences of zebrafish (Danio process, such as protein C (PROC, encoded by the proc gene) [21], rerio) from the NCBI database (D. rerio serpinb1, Accession no. NM_ plasmin [22], tissue plasminogen activator (tPA, encoded by the tpa 001002653; D. rerio serpinc1, Accession no. NM_182863; D. rerio ser- gene) and the urokinase plasminogen activator (uPA, encoded by the pind1, Accession no. NM_182880; D. rerio serpinf2b, Accession no NM_ upa gene) [23]. The complex formed by the combination of thrombin 001080010; and D. rerio serping1, Accession no. NM_001123285) were and the thrombomodulin (TM, encoded by the thbd gene)